Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
CLINICAL TOXICOLOGY 11( l), pp. 107-116 (1977)
Enhanced Polychlorinated Biphenyl Lesions in Moloney Leukemia Virus-Infected Mice
LOREN D. KOLLER, D.V.M., Ph.D.
*
National Institute of Environmental Health Sciences Research Triangle Park, North Carolina
INTRODUCTION Polychlorinated biphenyls (PCB) are potential environmental contaminants that are used in electrical capacitors and transformers, heat transfer systems, plasticizer applications, hydraulic lubricants, and other miscellaneous applications [l]. In 1968, the ingestion of K rice oil contaminated with PCB Kanechlor 400 (48% chlorine) produced the disease i'Yusho" in Japan [2]. The disease was characterized by increased eye discharge, follicular accentuation, acne-form eruption, sweating palms, weakness, and pigmentation of the skin and nails. Polychlorinated biphenyls are hepatotoxic in mammals [3-51 but produce edema formation in birds [3]. Since PCBs are hepatotoxic and neoplasia produced by Moloney leukemia virus (MLV) may involve the liver, the present study was devised to determine if different formulations of PCB may affect induction of neoplasia by MLV o r if MLV may affect the toxicity of PCB.
*Address reprint requests to Dr. Loren D. Koller, School of Veterinary Medicine-Oregon State University, Corvallis, Oregon 9733 1.
107 Copyright 0 1977 by Marcel Dekker, Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any meahs, electronic or mechanical, including photocopying. microfilming. and recording. or by any information storage and retrieval system, without permission in writing from the publlsher
KOLLER
108
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
MATERIALS AND METHODS Balb/c male mice 28 days old were used in the experiment. The animals were divided into 10 groups. Nine groups of 100 mice each received PCB Aroclor 1221, 1242, o r 1254 (Monsanto Co., St. Louis, Mo.) mixed in the feed (Ralston Purina Lab., St. Louis, Mo.) and 175 controls were given non-PCB-contaminated feed. The PCB formulations contained 21, 42, and 54% chlorine. The PCB-exposed groups received either 375, 37.5, o r 3.75 ppm of each Aroclor (1221, 1242, o r 1254) (Table 1). Twenty-five mice in each of the nine PCB groups were inoculated intraperitoneally (IP) a t 42 days of age with 0.3 ml of either 1 X loo, 1 x o r 1 X lo-' dilution of Moloney leukemia virus (MLV) (NCI, NIH by University Lab. Inc., Highland Park, N. J.), and the remaining 25 were inoculated IP with sterile physologic saline (Table 1). Fifty control mice (without PCB) were each inoculated IP a t 42 days of age with 0.3 ml of 1 X loo, 1 X lo-'*', o r 1 X lo-' dilution of MLV, and 25 mice received neither PCB nor MLV. The PCBs were fed for s i x months a t which time one-half of the animals in each group were killed. Each mouse was weighed and necropsied. The liver, spleen, and kidneys were weighed and placed in buffered 10% formalin. Feeding of the PCB-contaminated food was discontinued a t six months and mice that had been receiving 37.5 o r 3.75 ppm Aroclor 1254, 375 ppm Aroclor 1242, and no PCB were fed another three months of PCB-free diet. These animals were then killed, weighed, and necropsied. The liver, spleen, and kidneys were weighed and placed in 10% formalin. The mice remaining in the other PCB groups were killed, examined for g r o s s and microscopic lesions, and discarded after the s i x of months of feeding PCB since lesions had not occurred by that time. Microscopically, hepatic lesions were graded from + to ++++. The most prominent lesions of each grade were as follows: + was represented by mild centrolubular granular degeneration and necrosis of hepatocytes and a slight variation in cell size; ++ was determined by moderate vacuolar degeneration and necrosis of the centrolobular hepatocytes (Fig. 1) and several hepatocytes were enlarged; +++ changes consisted of s e v e r e centrolobular cytoplasmic vacuolar degeneration and necrosis of hepatocytes that often involved the entire lobule, marked variation in cell size, and nucleoli that were often prominent; ++++ lesions were characterized by extensive bile duct proliferation and periportal fibrosis, marked variation in cell size but most all were enlarged, necorsis of hepatocytes, and the nucleoli were distinct with margination of the chromatin (Fig. 2). Fat droplets occurred in some hepatocytes a t all grades of alteration, but there was never an abundant accumulation of fat. The spleen was considered to be altered by Moloney leukemia virus
None
3.75
37.5
375
3.75
375 37.5
1.74
2.13 1.71
1.82
1.63
1.98
3.11 1.97 1.98
1.79
1.96 1.92
1.75
2.05
2.85 2.06
2.07
6.01 2.47
9.46
6 mo.
1.76
2.43
1.57
1.96
9 mo.
1 x loo
~~~
1.73
1.88
1.83 1.91
1.93
2.79 1.98
2.08
5.79 2.60
8.82
6 mo.
1.80
2.56
1.84
1.86
9 mo.
1 x 10-1.5
~
1.66
1.70
1.99 1.84
1.74
3.27 2.01
1.96
6.05 2.55
9.10
6 mo.
1.82
2.41
1.79
1.91
9 mo.
ix 10-~
Mean liver weights are included f o r three exposures of PCB in which PCBs had been removed for three months after the six-month exposure (9 mo.). Aiialysis of variance revealed the increase in mean liver weights were highly significant ( p < 0.01) in each of the Aroclor 1254 groups and in the 375 ppm Aroclor 1242 group a t six months. There was a highly significant (~(0.01) decrease in mean liver weights when the PCB was removed from the diet f o r three months in the 37.5 ppm Aroclor 1254 groups and in the 375 Aroclor 1242 group. bDied before s i x months.
a
Control
1221
1242
1.87
2.01
9 mo.
2.11
5.48 2.57
375b 37.5
3.75
10.0
37 5
1254
6 mo.
ppm
PCB Aroclor
No virus
Dilution MLV
TABLE 1. Mean Liver Weights (gm) of Mice Given Three Different Aroclors of Polychlorinated Biphenyls f o r Six Months and Inoculated with Three Dilutions of Moloney Leukemia V i r u s a
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
(0
c 0
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
110
KOLLER
FIG. 1. Centrolobular necrosis of the liver that was considered a s a ++ PCB lesion. Notice the vacuolated hepatocytes.
FIG. 2. Bile duct proliferation and early fibrosis that was typical of a ++++ PCB lesion. Notice the variation in cell size and the prominent nucleoli in some of the enlarged nuclei.
POLYCHLORINATED BIPHENYL LESIONS
111
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
(neoplastic) when there was excessive hematopoesis, enlarged lymphoid nodules, o r neoplastic lymphocytes in the red o r white pulp. The spleen weights also increased when these alterations w ere evident. Since the spleen was considered to be the organ of origination of the neoplastic cells (lymphocytes), accumulations of neoplastic lymphocytes in the liver were considered as metastasis. RESULTS Many of the mice that received 375 ppm Aroclor 1254 died during the fifth and sixth month due to PCB toxicity. This dose of PCB was the only one that was toxic during the six-month feeding period. Those that died and were not inoculated with MLV had s e v e r e centrolobular to diffuse cytoplasmic vacuolar degeneration and necrosi s of the hepatocytes. Many hepatic cel l s were enlarged but t here was a marked variation of size throughout. The nucleoli were often prominent with margination of chromatin in cells that had enlarged nuclei. These histopathologic changes were considered to be +++. Those mice that received 375 ppm Aroclor 1254 and 1 X 10' o r 1 X lo-'.' MLV that had spleens affected by the vi rus had increased liver damage from +++ to ++++ (Table 2). Histologically, t here was usually extensive bile duct proliferation and periportal fibrosis of the liver. The hepatocytes varied considerably in size but most w ere enlarged. Necrosis of the cel l s was prominent and several contained hyalinized bodies. The nucleoli were distinct in the enlarged nuclei and the chromatin was marginated. These lesions w ere characteristic of a +++tliv er response to PCB (Fig. 2). Those l i vers from m i ce that did not exhibit splenic change w er e s i m i l a r to the nonvirus group and had +++ liv er change. Only eight mice survived for s i x months on the 375 ppm Aroclor 1254 diet. These mice had ++++ liver lesions and seven had splenic involvement. Mice given 37.5 ppm Aroclor 1254 without virus had + l i ver lesions characterized by a mild centrolobular granular degeneration and necrosis of hepatocytes and a slight variation in cell size. Those that w ere inoculated with MLV and had splenic involvement had ++ l i ver lesions (Table 2) represented by a moderate vacuolar degeneration and necrosis of the centrolobular hepatocytes (Fig. 1). Several hepatocytes were enlarged and variation in size was apparent. Liver lesions did not develop in the 3.75 ppm Aroclor 1254 and nonvirus group, but ++ l i ver lesions occurred when mice were inoculated with 1 X 10' vi rus and exhibited splenic alterations. Mice that received 375 ppm Aroclor 1242 without MLV had ++ l i ver lesions while those that w er e inoculated with 1 X 10' vi rus and had splenic involvement had +++ l i ver lesions (Table 2). A s i m i l a r effect
rime
37.5 3.75
-
++
-
-
+
-
-
-
++
++t
++ ++
++++C
+
+++
6 mo. ++++
9 mo.
-
+ t o +++
+ +
9 mo.
1 x loo
++++
6 mo.
No virus
-
++
+
+-I++
++++
6 mo.
-
-
++
-
-
-
+ +to +++
+
++++ +++ +
-
-
-
9 mo.
1 x 1 0 - ~o 6 mo.
9 mo.
1 x lo-l-s
No hepatic lesions. +, mild centrolobular degeneration and necrosis hepatocytes, slight variation in size hepatocytes. ++, moderate centrolobular degeneration and necrosis hepatocytes, s e v e r a l hepatocytes enlarged. +++, s e v e r e centrolobular degeneration and necrosis hepatocytes, marked variation in cell size, nucleoli prominent. ++++, bile duct proliferation, periportal fibrosis, marked variation in cell size, necrosis of hepatocytes, distinct nucleoli with margination of chromatin. bDied before s i x months. CIncrease in lesion severity f o r each virus-inoculated group occurred only in those mice that developed splenic lesions due to MLV.
Control
1221
375.0 37.5
1242
3.75 375.0
375.0 375.e 37.5 3.75
ppm
1254
PC B Aroclor
Dilution of MLV
TABLE 2. Severity of Hepatic Lesions in Mice Exposed to Three Different Aroclors of Polychlorinated Biphenyls and Inoculated with Three Dosages of Moloney Leukemia V i r u s a
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
+
F
tsl
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
POLYCHLORINATED BIPHENYL LESIONS
113
occurred at 37.5 ppm Aroclor 1242. Liver lesions did not o c c u r unless splenic lesions w e r e evident in the MLV-inoculated m i c e and then liver lesions developed. At s i x months, r e g a r d l e s s of the v i r u s dose, whenever there was evidence of neoplasia in the spleen by histopathology and increased spleen weight, the PCB l i v e r lesions w e r e increased in severity by one +. At nine months (three months after discontinuing P C B feed), m i c e in the 375 ppm Aroclor 1242 group without v i r u s did not manifest l i v e r lesions, but those inoculated with 1 X 10' MLV had l i v e r changes ranging from + to +++ (Table 2). Seventy-three percent had lesions; the majority (36%) had advanced lesions (+++). However in the 1 X 10- 1.5 MLV group, 71% had l i v e r lesions ranging from + to +++ but the majority (43%) had only mild lesions (+). Microscopically, alterations did not occur in the spleens n o r w e r e their weights increased in the virus-inoculated animals as w a s observed in the six-month animals. Apparently, the virus enhanced P C B l i v e r change, but did not produce neoplasia in the spleen as it had in other animals. The l i v e r s from mice given 37.5 and 3.75 ppm Aroclor 1254 for s i x months and then removed from i t for three months had lesions s i m i l a r to those a t six months, (Table 2), indicating that l i v e r damage from PCB was not reversible in the three-month period. However, l i v e r s from mice given 375 ppm Aroclor 1242 had ++ P C B lesions at s i x months, but lesions w e r e not present at nine months, indicating that the l i v e r had regenerated. The mice that lived f o r s i x months in the 375 ppm Aroclor 1254 group had the largest l i v e r s (Table 1). Those that died e a r l y also had l i v e r s that were markedly enlarged and that w e r e t h r e e t i m e s l a r g e r than control livers. The l i v e r weights in 37.5 and 3.75 groups decreased in size accordingly but remained l a r g e r than the controls. The l i v e r s were s m a l l e r in the 37.5 and 3.75 ppm groups at nine months than at s i x months, suggesting that the lesions had regressed. The PCBs did not produce hepatic neoplasia in the six-month feeding period n o r did MLV produce l i v e r pathology o t h e r than infrequent m e t a s t a s i s of neoplastic cells to the l i v e r from the spleen. DISCUSSION Hepatic toxicity produced by PCBs was enhanced by MLV. Hepatic lesions in the mice exposed to PCB f o r s i x months w e r e increased in severity by MLV only when the v i r u s produced evidence of neoplasia in the spleen. Regardless of the v i r u s dose, whenever t h e r e was a n i n c r e a s e in weight and histologic alteration of the spleen, the hepatic lesions were more pronounced. When MLV did not affect the spleen, the P C B l i v e r lesions w e r e s i m i l a r to those of non-virus-inoculated
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
114
KOLLER
mice. T h r e e months a f t e r removal from the P C B diet, hepatic lesions were again advanced by MLV. However, the spleens w e r e usually free of histologic evidence of neoplasia and t h e i r weight was comparable with spleens from non-virus-inoculated control mice. Liver weights corresponded with the dose of P C B given. The highe s t dose of Aroclor 1254 produced the l a r g e s t l i v e r s , and the l i v e r s decreased in s i z e as the dose diminished (Table 1). Only the highest dose of Aroclor 1242 (375 ppm) produced enlarged livers, while the other doses of 1242 and 1221 had no effect on l i v e r weight. Histopathology coincided with liver weights since hepatic lesions w e r e apparent only a t those doses that resulted in increased l i v e r weights. Liver weights decreased and returned n e a r the non-PCB-exposed l i v e r weights in those mice that w e r e removed from the P C B feed a t six months and were then fed a PCB-free diet f o r an additional t h r e e months. Histologically, the l i v e r s had recovered from the toxic hepatitis in the 375 ppm Aroclor 1242 group (Table 2), but hepatic lesions remained apparent in the 37.5 ppm Aroclor 1254 mice. P e r h a p s the residues of Aroclor 1254 are detoxified m o r e slowly by the l i v e r than those of Aroclors 1242 and 1221. However, since PCBs are s t o r e d in fat [6] and Aroclor 1254 a p p e a r s to be m o r e toxic than Aroclor 1221 and 1242 [ 51, s m a l l quantities of residues may be r e l e a s e d continuously from fat depots, prolonging complete detoxification by the liver. PCBs have promoted the induction of chemically induced neoplasms by benzene hexachloride in m i c e [7]. In the c u r r e n t study, PCBs did not promote the induction of neoplasia by MLV. Many environmental contaminants have been reported to be synergistic to infectious agents [8-131, but only two have been incriminated to activate a n oncogenic v i r u s [ 141. Several of those contaminants [ 15- 171, including PCBs [ 18, 191 ; are apparently immunosuppressive. Since oncogenic v i r u s e s are often activated in immunosuppressed hosts [20, 211, and P C B inc r e a s e d the pathogenecity of a nononcogenic v i r u s [8], it was anticipated that P C B would activate MLV. This did not o c c u r in o u r experiment. Perhaps the immunosuppression, if it did occur, w a s not sufficient to activate the virus. These results conform with a recent study [ 131 in which methylmercury chloride increased the susceptibility of m i c e to a nononcogenic (encephalomyocarditis) virus, but not to a n oncogenic (RLV) virus. The PCBs, 500 ppm Kanechlor 500 and 300 ppm Aroclor 1254, produced neoplastic changes in l i v e r s of mice when fed f o r eight and eleven months, respectively [7, 221. In the present study, neoplasia did not occur in m i c e fed 375 ppm P C B Aroclors 1254, 1242, and 1221 f o r s i x months. The results of this experiment demonstrated that an oncogenic v i r u s ( MLV) enhanced PCB-induced hepatic toxicity but conversely, P C B s did not appear to activate MLV. It was a l s o demonstrated that PCBproduced hepatic lesions (noncirrhotic) do regenerate a f t e r removal
POLYCHLORINATED BIPHENYL LESIONS
115
of PCB from the diet. To my knowledge, this is the f i r s t report to indicate that an oncogenic virus enhances toxicity of an environmental con tam inant.
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
SUMMARY The hepatic toxicity produced by polychlorinated biphenyls (PCB) was enhanced in mice that were inoculated with an oncogenic virus, Moloney leukemia virus (MLV). Whenever there was neoplastic involvement of the spleen by MLV, the hepatic lesions produced by PCB were more pronounced than in those of non-MLV inoculated mice. Mice were exposed to PCB Aroclors, 1254, 1242, and 1221 for s i x months. Aroclors 1254 and 1242 were hepatotoxic with Aroclor 1254 causing death. Aroclor 1221 did not affect the mice. Liver weights in mice that were fed PCBs for s i x months and then maintained on a PCB-free diet for an additional three months were comparable with those of non-PCB exposed mice. These results suggest that the PCB-produced hepatic lesions (noncirrhotic) regenerate after removal of PCB from the diet. Polychlorinated biphenyls did not affect (promote o r induce) the oncogenesis of MLV in this study. ACKNOWLEDGMENTS The author thanks Dr. J. A. Moore, Mrs. L. D. Lawson, and Mr. Z. McCoy for valuable assistance and Dr. Roger Peterson f o r statistical analysis. REFERENCES
[l] I. C. T. Nibet and A. F. Sarofim, Rates and routes of transport of PCB's in the environment, Environ. Health Persp., & 21 ( 1972). [2] M. Kuratuse, Y. Yoshimura, J. Matsuzaka et al., Epidemiologic study on Yusho, a poisoning caused by ingestion of rice oil contaminated with a commercial brand of polychlorinated biphenyls, Environ. Health Persp., 2, 119 (1972). [3] J. G. Vos, Toxicology of P C B ' s f o r mammals and f o r birds, Environ. Health Persp., 2, 105 (1972). [4] R. D. Kimbrough, R. E. Linder, and T. B Gaines, Morphological changes in livers of r a t s fed polychlorinated biphenyls, Arch. Environ. Health, 25, 354 (1972).
KOLLER
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
116
L. D. Koller and J. G. Zinkl, Pathology of polychlorinated biphenyls in rabbits, Am. J. Pathol., 3 363 (1973). A. Curley, J. W. Burse, M. E. Grim, et al., Polychlorinated biphenyls: Distribution and s t o r a g e in body fluids and tissues of Sherman r a t s , Environ. Res., 2, 481 (1971). I. Nobuyuki, H. Nagasaki, M. Arai, et al., Histopathologic studies on l i v e r tumorigenesis induced in m i c e by technical polychlorinated biphenyls and its promoting effect on l i v e r tumors induced by benzene hexachloride, J. Natl. C a n c e r Inst., 2,1637 ( 1973). M. Friend and D. 0. T r a i n e r , Polychlorinated biphenyl: Interaction with duck hepatitis virus, 9 -Science 9-170 1314 (1970). J. H. Gainer and T. W. P r y , Effects of a r s e n i c a l s on viral infections in mice, Am. J. Vet. Res., 33, 2299 (1972). J. H. Gainer, Increased m o r t a l i t r i n encephalomyocarditis v i r u s infected mice consumine: cobalt sulfate: T i s s u e concentration of cobalt, Am. J. Vet. Resr, 3 2607 (1972). G. A. Fairchild. J. Roan, and J. McCarroll. Atmospheric pollutants and the pathogenesis of viral r e s p i r a t o r y infection, Arch. Environ. Health, 25, 174 (1972). F. E. Hemphill, M. L. Kaeberle, and W. B. Buck, Lead suppression of mouse resistance to Salmonella typhimurium, Science,
172, 1031 (1971).
L. D. Koller, Methylmercury: Effect on oncogenic and non-oncotenic v i r u s e s in mice, J. Am. Vet. Med. ASSOC., 36, 1501 (1975). J. H. Gainer, Activation of the Rauscher leukemia v i r u s by metals, J. Natl. C a n c e r Inst., 3 609 (1973). L. D. Koller, Immunosuppression produced by lead, cadmium, and m e r c u r y , Am. J. Vet. Res., 3,1457 (1973). M. Wassermann, D. Wassermann, D. Kedar, et al., Immunological and detoxication interaction in p,p-DDT fed rabbits, Bull. Environ. Contam. Tox., 6, 426 (1971). L. D. Koller and S. Kovacic, Decreased antibody formation in mice exposed to lead, _ Nature, _ _ -250, 148 (1974). L. D. Koller and J. E. Thigpen, Reduction of antibody to pseudor a b i e s virus in polychlorinated biphenyl-exposed rabbits, Am. J. Vet. Res., 34, 1605 (1973). J. G. Vos ayd T. D. Roij, Immunosuppressive activity of a polychlorinated biphenyl preparation on the humoral immune response in guinea pigs, Toxicol. Appl. Pharmacol., 21, 549 (1972). M. S. Hirsch, P. H. Black, and M. R. Proffitt, Immunosuppression and oncogenic virus infections, Federation Proc., 30, 1852 (1971). W. S. Ceglowski and H. Friedman, Virus Tumorigenesis and Immunogenesis, Academic, New York, 1973, pp. 62-63, 131-133 R. D. Kimbrough and R. E. Linder, Induction of adenofibrosis and hepatomas of the l i v e r in Balb/dj mice by polychlorinated biphenyls (Aroclor 1254), J. Natl. C a n c e r Inst., 2, 547 (1974).
CLINICAL TOXICOLOGY 11( l), pp. 107-116 (1977)
Enhanced Polychlorinated Biphenyl Lesions in Moloney Leukemia Virus-Infected Mice
LOREN D. KOLLER, D.V.M., Ph.D.
*
National Institute of Environmental Health Sciences Research Triangle Park, North Carolina
INTRODUCTION Polychlorinated biphenyls (PCB) are potential environmental contaminants that are used in electrical capacitors and transformers, heat transfer systems, plasticizer applications, hydraulic lubricants, and other miscellaneous applications [l]. In 1968, the ingestion of K rice oil contaminated with PCB Kanechlor 400 (48% chlorine) produced the disease i'Yusho" in Japan [2]. The disease was characterized by increased eye discharge, follicular accentuation, acne-form eruption, sweating palms, weakness, and pigmentation of the skin and nails. Polychlorinated biphenyls are hepatotoxic in mammals [3-51 but produce edema formation in birds [3]. Since PCBs are hepatotoxic and neoplasia produced by Moloney leukemia virus (MLV) may involve the liver, the present study was devised to determine if different formulations of PCB may affect induction of neoplasia by MLV o r if MLV may affect the toxicity of PCB.
*Address reprint requests to Dr. Loren D. Koller, School of Veterinary Medicine-Oregon State University, Corvallis, Oregon 9733 1.
107 Copyright 0 1977 by Marcel Dekker, Inc All Rights Reserved Neither this work nor any part may be reproduced or transmitted in any form or by any meahs, electronic or mechanical, including photocopying. microfilming. and recording. or by any information storage and retrieval system, without permission in writing from the publlsher
KOLLER
108
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
MATERIALS AND METHODS Balb/c male mice 28 days old were used in the experiment. The animals were divided into 10 groups. Nine groups of 100 mice each received PCB Aroclor 1221, 1242, o r 1254 (Monsanto Co., St. Louis, Mo.) mixed in the feed (Ralston Purina Lab., St. Louis, Mo.) and 175 controls were given non-PCB-contaminated feed. The PCB formulations contained 21, 42, and 54% chlorine. The PCB-exposed groups received either 375, 37.5, o r 3.75 ppm of each Aroclor (1221, 1242, o r 1254) (Table 1). Twenty-five mice in each of the nine PCB groups were inoculated intraperitoneally (IP) a t 42 days of age with 0.3 ml of either 1 X loo, 1 x o r 1 X lo-' dilution of Moloney leukemia virus (MLV) (NCI, NIH by University Lab. Inc., Highland Park, N. J.), and the remaining 25 were inoculated IP with sterile physologic saline (Table 1). Fifty control mice (without PCB) were each inoculated IP a t 42 days of age with 0.3 ml of 1 X loo, 1 X lo-'*', o r 1 X lo-' dilution of MLV, and 25 mice received neither PCB nor MLV. The PCBs were fed for s i x months a t which time one-half of the animals in each group were killed. Each mouse was weighed and necropsied. The liver, spleen, and kidneys were weighed and placed in buffered 10% formalin. Feeding of the PCB-contaminated food was discontinued a t six months and mice that had been receiving 37.5 o r 3.75 ppm Aroclor 1254, 375 ppm Aroclor 1242, and no PCB were fed another three months of PCB-free diet. These animals were then killed, weighed, and necropsied. The liver, spleen, and kidneys were weighed and placed in 10% formalin. The mice remaining in the other PCB groups were killed, examined for g r o s s and microscopic lesions, and discarded after the s i x of months of feeding PCB since lesions had not occurred by that time. Microscopically, hepatic lesions were graded from + to ++++. The most prominent lesions of each grade were as follows: + was represented by mild centrolubular granular degeneration and necrosis of hepatocytes and a slight variation in cell size; ++ was determined by moderate vacuolar degeneration and necrosis of the centrolobular hepatocytes (Fig. 1) and several hepatocytes were enlarged; +++ changes consisted of s e v e r e centrolobular cytoplasmic vacuolar degeneration and necrosis of hepatocytes that often involved the entire lobule, marked variation in cell size, and nucleoli that were often prominent; ++++ lesions were characterized by extensive bile duct proliferation and periportal fibrosis, marked variation in cell size but most all were enlarged, necorsis of hepatocytes, and the nucleoli were distinct with margination of the chromatin (Fig. 2). Fat droplets occurred in some hepatocytes a t all grades of alteration, but there was never an abundant accumulation of fat. The spleen was considered to be altered by Moloney leukemia virus
None
3.75
37.5
375
3.75
375 37.5
1.74
2.13 1.71
1.82
1.63
1.98
3.11 1.97 1.98
1.79
1.96 1.92
1.75
2.05
2.85 2.06
2.07
6.01 2.47
9.46
6 mo.
1.76
2.43
1.57
1.96
9 mo.
1 x loo
~~~
1.73
1.88
1.83 1.91
1.93
2.79 1.98
2.08
5.79 2.60
8.82
6 mo.
1.80
2.56
1.84
1.86
9 mo.
1 x 10-1.5
~
1.66
1.70
1.99 1.84
1.74
3.27 2.01
1.96
6.05 2.55
9.10
6 mo.
1.82
2.41
1.79
1.91
9 mo.
ix 10-~
Mean liver weights are included f o r three exposures of PCB in which PCBs had been removed for three months after the six-month exposure (9 mo.). Aiialysis of variance revealed the increase in mean liver weights were highly significant ( p < 0.01) in each of the Aroclor 1254 groups and in the 375 ppm Aroclor 1242 group a t six months. There was a highly significant (~(0.01) decrease in mean liver weights when the PCB was removed from the diet f o r three months in the 37.5 ppm Aroclor 1254 groups and in the 375 Aroclor 1242 group. bDied before s i x months.
a
Control
1221
1242
1.87
2.01
9 mo.
2.11
5.48 2.57
375b 37.5
3.75
10.0
37 5
1254
6 mo.
ppm
PCB Aroclor
No virus
Dilution MLV
TABLE 1. Mean Liver Weights (gm) of Mice Given Three Different Aroclors of Polychlorinated Biphenyls f o r Six Months and Inoculated with Three Dilutions of Moloney Leukemia V i r u s a
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
(0
c 0
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
110
KOLLER
FIG. 1. Centrolobular necrosis of the liver that was considered a s a ++ PCB lesion. Notice the vacuolated hepatocytes.
FIG. 2. Bile duct proliferation and early fibrosis that was typical of a ++++ PCB lesion. Notice the variation in cell size and the prominent nucleoli in some of the enlarged nuclei.
POLYCHLORINATED BIPHENYL LESIONS
111
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
(neoplastic) when there was excessive hematopoesis, enlarged lymphoid nodules, o r neoplastic lymphocytes in the red o r white pulp. The spleen weights also increased when these alterations w ere evident. Since the spleen was considered to be the organ of origination of the neoplastic cells (lymphocytes), accumulations of neoplastic lymphocytes in the liver were considered as metastasis. RESULTS Many of the mice that received 375 ppm Aroclor 1254 died during the fifth and sixth month due to PCB toxicity. This dose of PCB was the only one that was toxic during the six-month feeding period. Those that died and were not inoculated with MLV had s e v e r e centrolobular to diffuse cytoplasmic vacuolar degeneration and necrosi s of the hepatocytes. Many hepatic cel l s were enlarged but t here was a marked variation of size throughout. The nucleoli were often prominent with margination of chromatin in cells that had enlarged nuclei. These histopathologic changes were considered to be +++. Those mice that received 375 ppm Aroclor 1254 and 1 X 10' o r 1 X lo-'.' MLV that had spleens affected by the vi rus had increased liver damage from +++ to ++++ (Table 2). Histologically, t here was usually extensive bile duct proliferation and periportal fibrosis of the liver. The hepatocytes varied considerably in size but most w ere enlarged. Necrosis of the cel l s was prominent and several contained hyalinized bodies. The nucleoli were distinct in the enlarged nuclei and the chromatin was marginated. These lesions w ere characteristic of a +++tliv er response to PCB (Fig. 2). Those l i vers from m i ce that did not exhibit splenic change w er e s i m i l a r to the nonvirus group and had +++ liv er change. Only eight mice survived for s i x months on the 375 ppm Aroclor 1254 diet. These mice had ++++ liver lesions and seven had splenic involvement. Mice given 37.5 ppm Aroclor 1254 without virus had + l i ver lesions characterized by a mild centrolobular granular degeneration and necrosis of hepatocytes and a slight variation in cell size. Those that w ere inoculated with MLV and had splenic involvement had ++ l i ver lesions (Table 2) represented by a moderate vacuolar degeneration and necrosis of the centrolobular hepatocytes (Fig. 1). Several hepatocytes were enlarged and variation in size was apparent. Liver lesions did not develop in the 3.75 ppm Aroclor 1254 and nonvirus group, but ++ l i ver lesions occurred when mice were inoculated with 1 X 10' vi rus and exhibited splenic alterations. Mice that received 375 ppm Aroclor 1242 without MLV had ++ l i ver lesions while those that w er e inoculated with 1 X 10' vi rus and had splenic involvement had +++ l i ver lesions (Table 2). A s i m i l a r effect
rime
37.5 3.75
-
++
-
-
+
-
-
-
++
++t
++ ++
++++C
+
+++
6 mo. ++++
9 mo.
-
+ t o +++
+ +
9 mo.
1 x loo
++++
6 mo.
No virus
-
++
+
+-I++
++++
6 mo.
-
-
++
-
-
-
+ +to +++
+
++++ +++ +
-
-
-
9 mo.
1 x 1 0 - ~o 6 mo.
9 mo.
1 x lo-l-s
No hepatic lesions. +, mild centrolobular degeneration and necrosis hepatocytes, slight variation in size hepatocytes. ++, moderate centrolobular degeneration and necrosis hepatocytes, s e v e r a l hepatocytes enlarged. +++, s e v e r e centrolobular degeneration and necrosis hepatocytes, marked variation in cell size, nucleoli prominent. ++++, bile duct proliferation, periportal fibrosis, marked variation in cell size, necrosis of hepatocytes, distinct nucleoli with margination of chromatin. bDied before s i x months. CIncrease in lesion severity f o r each virus-inoculated group occurred only in those mice that developed splenic lesions due to MLV.
Control
1221
375.0 37.5
1242
3.75 375.0
375.0 375.e 37.5 3.75
ppm
1254
PC B Aroclor
Dilution of MLV
TABLE 2. Severity of Hepatic Lesions in Mice Exposed to Three Different Aroclors of Polychlorinated Biphenyls and Inoculated with Three Dosages of Moloney Leukemia V i r u s a
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
+
F
tsl
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
POLYCHLORINATED BIPHENYL LESIONS
113
occurred at 37.5 ppm Aroclor 1242. Liver lesions did not o c c u r unless splenic lesions w e r e evident in the MLV-inoculated m i c e and then liver lesions developed. At s i x months, r e g a r d l e s s of the v i r u s dose, whenever there was evidence of neoplasia in the spleen by histopathology and increased spleen weight, the PCB l i v e r lesions w e r e increased in severity by one +. At nine months (three months after discontinuing P C B feed), m i c e in the 375 ppm Aroclor 1242 group without v i r u s did not manifest l i v e r lesions, but those inoculated with 1 X 10' MLV had l i v e r changes ranging from + to +++ (Table 2). Seventy-three percent had lesions; the majority (36%) had advanced lesions (+++). However in the 1 X 10- 1.5 MLV group, 71% had l i v e r lesions ranging from + to +++ but the majority (43%) had only mild lesions (+). Microscopically, alterations did not occur in the spleens n o r w e r e their weights increased in the virus-inoculated animals as w a s observed in the six-month animals. Apparently, the virus enhanced P C B l i v e r change, but did not produce neoplasia in the spleen as it had in other animals. The l i v e r s from mice given 37.5 and 3.75 ppm Aroclor 1254 for s i x months and then removed from i t for three months had lesions s i m i l a r to those a t six months, (Table 2), indicating that l i v e r damage from PCB was not reversible in the three-month period. However, l i v e r s from mice given 375 ppm Aroclor 1242 had ++ P C B lesions at s i x months, but lesions w e r e not present at nine months, indicating that the l i v e r had regenerated. The mice that lived f o r s i x months in the 375 ppm Aroclor 1254 group had the largest l i v e r s (Table 1). Those that died e a r l y also had l i v e r s that were markedly enlarged and that w e r e t h r e e t i m e s l a r g e r than control livers. The l i v e r weights in 37.5 and 3.75 groups decreased in size accordingly but remained l a r g e r than the controls. The l i v e r s were s m a l l e r in the 37.5 and 3.75 ppm groups at nine months than at s i x months, suggesting that the lesions had regressed. The PCBs did not produce hepatic neoplasia in the six-month feeding period n o r did MLV produce l i v e r pathology o t h e r than infrequent m e t a s t a s i s of neoplastic cells to the l i v e r from the spleen. DISCUSSION Hepatic toxicity produced by PCBs was enhanced by MLV. Hepatic lesions in the mice exposed to PCB f o r s i x months w e r e increased in severity by MLV only when the v i r u s produced evidence of neoplasia in the spleen. Regardless of the v i r u s dose, whenever t h e r e was a n i n c r e a s e in weight and histologic alteration of the spleen, the hepatic lesions were more pronounced. When MLV did not affect the spleen, the P C B l i v e r lesions w e r e s i m i l a r to those of non-virus-inoculated
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
114
KOLLER
mice. T h r e e months a f t e r removal from the P C B diet, hepatic lesions were again advanced by MLV. However, the spleens w e r e usually free of histologic evidence of neoplasia and t h e i r weight was comparable with spleens from non-virus-inoculated control mice. Liver weights corresponded with the dose of P C B given. The highe s t dose of Aroclor 1254 produced the l a r g e s t l i v e r s , and the l i v e r s decreased in s i z e as the dose diminished (Table 1). Only the highest dose of Aroclor 1242 (375 ppm) produced enlarged livers, while the other doses of 1242 and 1221 had no effect on l i v e r weight. Histopathology coincided with liver weights since hepatic lesions w e r e apparent only a t those doses that resulted in increased l i v e r weights. Liver weights decreased and returned n e a r the non-PCB-exposed l i v e r weights in those mice that w e r e removed from the P C B feed a t six months and were then fed a PCB-free diet f o r an additional t h r e e months. Histologically, the l i v e r s had recovered from the toxic hepatitis in the 375 ppm Aroclor 1242 group (Table 2), but hepatic lesions remained apparent in the 37.5 ppm Aroclor 1254 mice. P e r h a p s the residues of Aroclor 1254 are detoxified m o r e slowly by the l i v e r than those of Aroclors 1242 and 1221. However, since PCBs are s t o r e d in fat [6] and Aroclor 1254 a p p e a r s to be m o r e toxic than Aroclor 1221 and 1242 [ 51, s m a l l quantities of residues may be r e l e a s e d continuously from fat depots, prolonging complete detoxification by the liver. PCBs have promoted the induction of chemically induced neoplasms by benzene hexachloride in m i c e [7]. In the c u r r e n t study, PCBs did not promote the induction of neoplasia by MLV. Many environmental contaminants have been reported to be synergistic to infectious agents [8-131, but only two have been incriminated to activate a n oncogenic v i r u s [ 141. Several of those contaminants [ 15- 171, including PCBs [ 18, 191 ; are apparently immunosuppressive. Since oncogenic v i r u s e s are often activated in immunosuppressed hosts [20, 211, and P C B inc r e a s e d the pathogenecity of a nononcogenic v i r u s [8], it was anticipated that P C B would activate MLV. This did not o c c u r in o u r experiment. Perhaps the immunosuppression, if it did occur, w a s not sufficient to activate the virus. These results conform with a recent study [ 131 in which methylmercury chloride increased the susceptibility of m i c e to a nononcogenic (encephalomyocarditis) virus, but not to a n oncogenic (RLV) virus. The PCBs, 500 ppm Kanechlor 500 and 300 ppm Aroclor 1254, produced neoplastic changes in l i v e r s of mice when fed f o r eight and eleven months, respectively [7, 221. In the present study, neoplasia did not occur in m i c e fed 375 ppm P C B Aroclors 1254, 1242, and 1221 f o r s i x months. The results of this experiment demonstrated that an oncogenic v i r u s ( MLV) enhanced PCB-induced hepatic toxicity but conversely, P C B s did not appear to activate MLV. It was a l s o demonstrated that PCBproduced hepatic lesions (noncirrhotic) do regenerate a f t e r removal
POLYCHLORINATED BIPHENYL LESIONS
115
of PCB from the diet. To my knowledge, this is the f i r s t report to indicate that an oncogenic virus enhances toxicity of an environmental con tam inant.
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
SUMMARY The hepatic toxicity produced by polychlorinated biphenyls (PCB) was enhanced in mice that were inoculated with an oncogenic virus, Moloney leukemia virus (MLV). Whenever there was neoplastic involvement of the spleen by MLV, the hepatic lesions produced by PCB were more pronounced than in those of non-MLV inoculated mice. Mice were exposed to PCB Aroclors, 1254, 1242, and 1221 for s i x months. Aroclors 1254 and 1242 were hepatotoxic with Aroclor 1254 causing death. Aroclor 1221 did not affect the mice. Liver weights in mice that were fed PCBs for s i x months and then maintained on a PCB-free diet for an additional three months were comparable with those of non-PCB exposed mice. These results suggest that the PCB-produced hepatic lesions (noncirrhotic) regenerate after removal of PCB from the diet. Polychlorinated biphenyls did not affect (promote o r induce) the oncogenesis of MLV in this study. ACKNOWLEDGMENTS The author thanks Dr. J. A. Moore, Mrs. L. D. Lawson, and Mr. Z. McCoy for valuable assistance and Dr. Roger Peterson f o r statistical analysis. REFERENCES
[l] I. C. T. Nibet and A. F. Sarofim, Rates and routes of transport of PCB's in the environment, Environ. Health Persp., & 21 ( 1972). [2] M. Kuratuse, Y. Yoshimura, J. Matsuzaka et al., Epidemiologic study on Yusho, a poisoning caused by ingestion of rice oil contaminated with a commercial brand of polychlorinated biphenyls, Environ. Health Persp., 2, 119 (1972). [3] J. G. Vos, Toxicology of P C B ' s f o r mammals and f o r birds, Environ. Health Persp., 2, 105 (1972). [4] R. D. Kimbrough, R. E. Linder, and T. B Gaines, Morphological changes in livers of r a t s fed polychlorinated biphenyls, Arch. Environ. Health, 25, 354 (1972).
KOLLER
Clinical Toxicology Downloaded from informahealthcare.com by McMaster University on 11/11/14 For personal use only.
116
L. D. Koller and J. G. Zinkl, Pathology of polychlorinated biphenyls in rabbits, Am. J. Pathol., 3 363 (1973). A. Curley, J. W. Burse, M. E. Grim, et al., Polychlorinated biphenyls: Distribution and s t o r a g e in body fluids and tissues of Sherman r a t s , Environ. Res., 2, 481 (1971). I. Nobuyuki, H. Nagasaki, M. Arai, et al., Histopathologic studies on l i v e r tumorigenesis induced in m i c e by technical polychlorinated biphenyls and its promoting effect on l i v e r tumors induced by benzene hexachloride, J. Natl. C a n c e r Inst., 2,1637 ( 1973). M. Friend and D. 0. T r a i n e r , Polychlorinated biphenyl: Interaction with duck hepatitis virus, 9 -Science 9-170 1314 (1970). J. H. Gainer and T. W. P r y , Effects of a r s e n i c a l s on viral infections in mice, Am. J. Vet. Res., 33, 2299 (1972). J. H. Gainer, Increased m o r t a l i t r i n encephalomyocarditis v i r u s infected mice consumine: cobalt sulfate: T i s s u e concentration of cobalt, Am. J. Vet. Resr, 3 2607 (1972). G. A. Fairchild. J. Roan, and J. McCarroll. Atmospheric pollutants and the pathogenesis of viral r e s p i r a t o r y infection, Arch. Environ. Health, 25, 174 (1972). F. E. Hemphill, M. L. Kaeberle, and W. B. Buck, Lead suppression of mouse resistance to Salmonella typhimurium, Science,
172, 1031 (1971).
L. D. Koller, Methylmercury: Effect on oncogenic and non-oncotenic v i r u s e s in mice, J. Am. Vet. Med. ASSOC., 36, 1501 (1975). J. H. Gainer, Activation of the Rauscher leukemia v i r u s by metals, J. Natl. C a n c e r Inst., 3 609 (1973). L. D. Koller, Immunosuppression produced by lead, cadmium, and m e r c u r y , Am. J. Vet. Res., 3,1457 (1973). M. Wassermann, D. Wassermann, D. Kedar, et al., Immunological and detoxication interaction in p,p-DDT fed rabbits, Bull. Environ. Contam. Tox., 6, 426 (1971). L. D. Koller and S. Kovacic, Decreased antibody formation in mice exposed to lead, _ Nature, _ _ -250, 148 (1974). L. D. Koller and J. E. Thigpen, Reduction of antibody to pseudor a b i e s virus in polychlorinated biphenyl-exposed rabbits, Am. J. Vet. Res., 34, 1605 (1973). J. G. Vos ayd T. D. Roij, Immunosuppressive activity of a polychlorinated biphenyl preparation on the humoral immune response in guinea pigs, Toxicol. Appl. Pharmacol., 21, 549 (1972). M. S. Hirsch, P. H. Black, and M. R. Proffitt, Immunosuppression and oncogenic virus infections, Federation Proc., 30, 1852 (1971). W. S. Ceglowski and H. Friedman, Virus Tumorigenesis and Immunogenesis, Academic, New York, 1973, pp. 62-63, 131-133 R. D. Kimbrough and R. E. Linder, Induction of adenofibrosis and hepatomas of the l i v e r in Balb/dj mice by polychlorinated biphenyls (Aroclor 1254), J. Natl. C a n c e r Inst., 2, 547 (1974).
